Abstract
The reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been widely used to determine gene functions in Laodelphax striatellus (Fallén) (small brown planthopper). Selection of suitable reference gene(s) for normalizations of RT-qPCR data is critical for reliable results. To date, reports on identification of suitable L. striatellus reference genes are still very limited. L. striatellus is a destructive rice pest and it can transmit multiple viruses, including Rice black-streaked dwarf virus (RBSDV), Rice stripe virus (RSV), and Maize rough dwarf virus (MRDV), to many important cereal crops worldwide. In this study, we examined the stablity of seven selected candidate reference genes in L. striatellus at different developmental stages, in different tissues, in RBSDV- or RSV-infected L. striatellus or in RBSDV-infected and Lssynaptojanin 1 (LsSYNJ1)-silenced L. striatellus. The RT-qPCR data representing individual candidate genes were analyzed using five different methods: the delta Ct method, geNorm, NormFinder, BestKeeper, and the RefFinder algorithm, respectively. The most stable reference gene for the specific condition was selected according to a comprehensive analysis using the RefFinder method. Ribosomal protein L5 (LsRPL5) and LsRPL8 are the most stably expressed genes in L. striatellus at different developmental stages. Alpha-1-tubulin (Lsα-TUB) is the most stably expressed reference gene in different tissues of RBSDV viruliferous (RBSDV-V) or non-viruliferous (RBSDV-NV) L. striatellus. LsRPL8 is the most stably expressed reference gene in RBSDV-V or RSV viruliferous (RSV-V) L. striatellus, while beta-tubulin (Lsβ-TUB) is the most stably expressed reference gene in RBSDV-V and LsSYNJ1-silenced L. striatellus. The selected reference genes were further investigated during analyses of RBSDV P5-1 and P10 gene expression in different tissues from RBSDV-V or RBSDV-NV L. striatellus. The stably expressed reference genes identified in this study will benefit future gene function studies using L. striatellus.
Highlights
Laodelphax striatellus (Fallén), Order: Hemiptera, Family: Delphacidae, is an important agricultural pest which attacks plants by sucking sap from pholem with its stylet.L. striatellus is an important insect vector for many plant viruses, including Rice black-streaked dwarf virus (RBSDV), Rice stripe virus (RSV), and Maize rough dwarf virus (MRDV) [1,2,3]
We evaluated LsACT, Lsα-TUB, Lsβ-TUB, LsGAPDH, LsRPL5, LsRPL8, and Ls18S rRNA for their usefulness as reference genes through reverse transcription quantitative polymerase chain reaction (RT-qPCR) using L. striatellus at different developmental stages, infected or not infected with a virus, in different L. striatellus tissues, and in RBSDV-infected and Lssynaptojanin 1 (LsSYNJ1)-silenced L. striatellus
Analysis of the RT-qPCR data using the Bestkeeper algorithm showed that Lsβ-TUB was the most stably expressed gene in various tissues from RBSDV-NV L. striatellus
Summary
Laodelphax striatellus (Fallén) (small brown planthopper), Order: Hemiptera, Family: Delphacidae, is an important agricultural pest which attacks plants by sucking sap from pholem with its stylet. Previous studies have selected some housekeeping genes as reference genes to normalize RT-qPCR data in L. striatellus. Ribosomal protein S11 (RPS11) were reported to be the most suitable reference genes in different Nilaparvata lugens (brown planthopper) tissues and at different developmental stages [24]. For Mal de Río Cuarto virus (MRCV)-infected Delphacodes kuscheli (planthopper), polyubiquitin C (UBI), ribosomal protein S18 (RPS18) and actin (ACT) were found to be the most suitable RT-qPCR reference genes [26]. Because a suitable reference gene in one insect may not be the same as in other insects, or in the same insect under different experimental conditions, the suitability of individual housekeeping genes for RT-qPCR data normalization must be checked under specific conditions prior to use. The selected suitable reference genes described here should benefit gene function studies in L.striatellus
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